A great deal of effort has been directed towards developing nonparenteral (needle-free) alternatives to traditional vaccine delivery. Nonparenteral vaccines offer a number of potential advantages over traditional vaccines including 1) the potential to confer mucosal as well as systemic immunity, 2) increased stability, 3) increased shelf-life, 4) elimination of needles and the need for specially trained healthcare specialists to administer vaccines, and 5) potentially lower costs. One such approach, transcutaneous immunization (TCI), is a non-invasive, safe method of delivering antigens directly onto bare skin. Immunization is achieved by direct topical application of a vaccine antigen. Despite the attractiveness of TCI, the technology is limited by the relative inefficiency of transport of large molecular weight vaccine antigens across intact skin. Recent innovations in transdermal delivery of drugs, including chemical enhancers, electricity, ultrasound, and microneedles, demonstrate the feasibility of large-molecule transport through the skin's permeation- barrier, specifically the stratum corneum. This outer layer of the skin is composed of tightly packed lipid molecules and the dense, crystalline arrangement of these lipids creates the essential barrier to prevent water loss and pathogen entry. Recent evidence has shown that this barrier can be overcome by properly structured nano-sized particles (nanocarriers). This proposal will compare three different nanocarriers (temperature-responsive hollow nanospheres, nanohydrogels, and star copolymers) for the ability to incorporate a model vaccine antigen and deliver that antigen through the stratum corneum to immune- responsive cells in the epidermis. The specialized assembly of each type of nanocarrier gives each unique properties and different interactions within the lipid channels of the stratum corneum. The use of nanocarriers for vaccine delivery is a platform technology, applicable to delivery of a variety of existing and potential vaccines. For the purposes of this proposal, we will utilize two different proteins: 1) Bovine Serum Albumin that has been fluorescently labeled to monitor incorporation and permeation of a macromolecular antigen, and 2) F1-V, a vaccine antigen from Yersinia pestis, the causative agent of plague, which we and others have shown to protect against aerosol challenge with virulent Y.pestis. The proposed studies will address important questions in vaccine delivery by application of nanotechnology through the exploitation of the novel properties of nanocarriers. The findings of these studies will be broadly applicable to a variety of vaccines and therapeutics and will further highlight the important role of nanotechnology in science and medicine.